Добірка наукової літератури з теми "Biotin transporter; Staphylococcus aureus"

Biotin protein ligase (BPL) inhibitors are a novel class of antibacterial that target clinically important methicillin-resistant Staphylococcus aureus (S. aureus). In S. aureus, BPL is a bifunctional protein responsible for enzymatic biotinylation of two biotin-dependent enzymes, as well as serving as a transcriptional repressor that controls biotin synthesis and import. In this report, we investigate the mechanisms of action and resistance for a potent anti-BPL, an antibacterial compound, biotinyl-acylsulfamide adenosine (BASA). We show that BASA acts by both inhibiting the enzymatic activity of BPL in vitro, as well as functioning as a transcription co-repressor. A low spontaneous resistance rate was measured for the compound (<10−9) and whole-genome sequencing of strains evolved during serial passaging in the presence of BASA identified two discrete resistance mechanisms. In the first, deletion of the biotin-dependent enzyme pyruvate carboxylase is proposed to prioritize the utilization of bioavailable biotin for the essential enzyme acetyl-CoA carboxylase. In the second, a D200E missense mutation in BPL reduced DNA binding in vitro and transcriptional repression in vivo. We propose that this second resistance mechanism promotes bioavailability of biotin by derepressing its synthesis and import, such that free biotin may outcompete the inhibitor for binding BPL. This study provides new insights into the molecular mechanisms governing antibacterial activity and resistance of BPL inhibitors in S. aureus.

ABSTRACT Increased production of penicillin-binding protein PBP 4 is known to increase peptidoglycan cross-linking and contributes to methicillin resistance in Staphylococcus aureus. The pbp4gene shares a 400-nucleotide intercistronic region with the divergently transcribed abcA gene, encoding an ATP-binding cassette transporter of unknown function. Our study revealed that methicillin stimulated abcA transcription but had no effects onpbp4 transcription. Analysis of abcA expression in mutants defective for global regulators showed that abcAis under the control of agr. Insertional inactivation ofabcA by an erythromycin resistance determinant did not influence pbp4 transcription, nor did it alter resistance to methicillin and other cell wall-directed antibiotics. However,abcA mutants showed spontaneous partial lysis on plates containing subinhibitory concentrations of methicillin due to increased spontaneous autolysis. Since the autolytic zymograms of cell extracts were identical in mutants and parental strains, we postulate an indirect role of AbcA in control of autolytic activities and in protection of the cells against methicillin.

ABSTRACT Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria. Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in Staphylococcus aureus, MdeA (multidrug efflux A). MdeA was identified from screening an S. aureus open reading frame expression library for resistance to antibiotic compounds. When overexpressed, MdeA confers resistance on S. aureus to a range of quaternary ammonium compounds and antibiotics, but not fluoroquinolones. MdeA is a 52-kDa protein with 14 predicted transmembrane segments. It belongs to the major facilitator superfamily and is most closely related, among known efflux proteins, to LmrB of Bacillus subtilis and EmrB of Escherichia coli. Overexpression of mdeA in S. aureus reduced ethidium bromide uptake and enhanced its efflux, which could be inhibited by reserpine and abolished by an uncoupler. The mdeA promoter was identified by primer extension. Spontaneous mutants selected for increased resistance to an MdeA substrate had undergone mutations in the promoter for mdeA, and their mdeA transcription levels were increased by as much as 15-fold. The mdeA gene was present in the genomes of all six strains of S. aureus examined. Uncharacterized homologs of MdeA were present elsewhere in the S. aureus genome, but their overexpression did not mediate resistance to the antibacterials tested. However, MdeA homologs were identified in other bacteria, including Bacillus anthracis, some of which were shown to be functional orthologs of MdeA.

The rates of infection by community-acquired multi-drug resistant Staphylococcus aureus have risen dramatically over fifteen years in the United States. Community-acquired multi-drug resistant Staphylococcus aureus is responsible for rapidly progressive diseases, including necrotizing pneumonia, severe sepsis, and necrotizing fasciitis. Consequently, novel antibacterial strategies are needed to combat the rising antibiotic resistance seen in community-acquired multi-drug resistant strains. We have screened the Nebraska Transposon Mutant Library for MRSA strains that are either susceptible or resistant to methanol extracts of Centaurea nigrescens leaves and flowers. 10 strains containing mutations affecting transporter proteins were identified as having either significant resistance or susceptibility to Centaurea extract. Insertions in two different drug efflux transporter families have been identified. The EmrB/QacA drug resistance transporter subfamily is a multi-drug efflux pump responsible for the export of toxic molecules from bacteria and yeast. The ABC transporters are involved in drug import and export. These results confirm the effectiveness of the screen as a means for identifying drug-resistance genes affected by the C. nigrescens methanolic extract and suggest a role for drug efflux proteins in the resistance of S. aureus community-acquired multi-drug resistant Staphylococcus aureus to antibacterial plant metabolites

ABSTRACT The multidrug transporter NorA contributes to the resistance ofStaphylococcus aureus to fluoroquinolone antibiotics by promoting their active extrusion from the cell. Previous studies with the alkaloid reserpine, the first identified inhibitor of NorA, indicate that the combination of a chemical NorA inhibitor with a fluoroquinolone could improve the efficacy of this class of antibiotics. Since reserpine is toxic to humans at the concentrations required to inhibit NorA, we sought to identify new inhibitors of NorA that may be used in a clinical setting. Screening of a chemical library yielded a number of structurally diverse inhibitors of NorA that were more potent than reserpine. The new inhibitors act in a synergistic manner with the most widely used fluoroquinolone, ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. Furthermore, the inhibitors dramatically suppress the emergence of ciprofloxacin-resistant S. aureus upon in vitro selection with this drug. Some of these new inhibitors, or their derivatives, may prove useful for augmentation of the antibacterial activities of fluoroquinolones in the clinical setting.

ABSTRACT From a mass-excised Staphylococcus aureus λZapII expression library, we cloned an operon encoding a novel ABC transporter with significant homology to bacterial siderophore transporter systems. The operon encodes four genes designatedsstA, -B, -C, and -Dencoding two putative cytoplasmic membrane proteins (sstAand sstB), an ATPase (sstC), and a membrane-bound 38-kDa lipoprotein (sstD). Thesst operon is preceded by two putative Fur boxes, which indicated that expression of the sst operon was likely to be iron dependent. SstD was overexpressed inEscherichia coli, purified by Triton X-114 phase partitioning, and used to generate monospecific antisera in rats. Immunoblotting studies located SstD in the membrane fraction ofS. aureus and showed that expression of the lipoprotein was reduced under iron-rich growth conditions. Triton X-114 partitioning studies on isolated membranes provided additional biochemical evidence that SstD in S. aureus is a lipoprotein. Immunoreactive polypeptides of approximately 38 kDa were detected in a wide range of staphylococcal species, but no antigenic homolog was detected inBacillus subtilis. Expression of SstD in vivo was confirmed by immunoblotting studies with S. aureus recovered from a rat intraperitoneal chamber implant model. To further define the contribution of SstD in promoting growth of S. aureus in vitro and in vivo, we used antisense RNA technology to modulate expression of SstD. Expression of antisense sstD RNA inS. aureus resulted in a decrease in SstD expression under both iron-rich and iron-restricted growth conditions. However, this reduction in SstD levels did not affect the growth of S. aureus in vitro in an iron-limited growth medium or when grown in an intraperitoneal rat chamber implant model in vivo.

Abstract S. aureus has 16 predicted two-component systems (TCS) that respond to a range of environmental stimuli, and allow for adaptation to stresses. Of these 16, three have no known function, and are not homologous to any other TCS found in closely related organisms. NsaRS is one such element, and belongs to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the Firmicutes. The regulators are defined by a small sensing domain within their histidine kinase, suggesting that they do not sense external signals, but stress in or at the membrane. Our characterization of NsaRS in this work reveals that, as with other IM-HK TCS, it responds to cell-envelope damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, CCCP and penicillin G. Additionally; we reveal that NsaRS regulates a downstream transporter, NsaAB, during nisin-induced stress. Phenotypically, nsaS mutants display a 200-fold decreased ability to develop resistance to another cell-wall targeting antibiotic, bacitracin. Microarray analysis reveals the transcription of 245 genes is altered in a nsaS mutant, with the vast majority down-regulated. Included within this list are genes involved in transport, drug-resistance, cell-envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using ICP-MS, a decrease in intracellular divalent metal ions was observed in an nsaS mutant, when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants also have alterations in cell-envelope structure. Finally, a variety of virulence related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus NsaRS is important in sensing cell wall damage in S. aureus, and functions to reprogram gene expression to modify cell-envelope architecture, facilitating adaptation and survival. Interestingly, in our microarray analysis, we observed a more than 30-fold decrease in transcription of an ABC transporter, SACOL2525/2526, in the nsaS mutant. This transporter bears strong homology to nsaAB, and is currently uncharacterized. Exploration of the role of SACOL2525/2526 revealed that, along with NsaRS, it too responds to cell-envelope damaging antibiotics. Specifically, its expression was induced by phosphomycin, daptomycin, penicillin G, ampicillin, oxacillin, D-cycloserine and CCCP. Mutation of this transporter results in increased sensitivity to the antibacterial agent daptomycin, and decreased sensitivity to free fatty acids. These findings are perhaps explained by altered membrane fluidity in the mutant strain, as the transporter null-strain is more readily killed in the presence of organic solvents, such as toluene. In addition, SACOL2525/2526 mutants have a decreased ability to form spontaneous mutants in response to several other peptidoglycan synthesis targeting antibiotics, suggesting a role for SACOL2525/2526 in antibiotic resistance. Inactivation of this transporter alters the cell envelope, and produces similar effects to those observed with the nsaS mutant, with increased capsule production, that may provide resistance to lysostaphin. Interestingly, the nsaS microarray revealed that this TCS negatively regulates only 34 genes, including 6 out of the 10 major secreted proteases. Despite a number of reports in the literature describing these enzymes as virulence factors, the data is often conflicting. Therefore, the contribution of proteases to CA-MRSA pathogenesis was investigated, by constructing a strain lacking all 10 extracellular protease genes. Analysis of this strain using murine models of infection reveals secreted proteases significantly impact virulence in both localized and systemic infections. Additionally, inactivation of these enzymes strongly influences survival in whole human blood, and increases sensitivity to antimicrobial peptides. Using a proteomics approach, we demonstrate that the contribution of secreted proteases to pathogenicity is related to differential processing of a large number of surface-associated virulence factors and secreted toxins. Collectively these findings provide a unique insight into the role of secreted proteases in CA-MRSA infections.

ECF transporters are a group of newly defined ABC-like modular transporters and they are composed of three main elements: 1) a high-affinity membrane-embedded substrate binding protein (S component), 2) a membrane-spanning protein (T component), and 3) two identical or homologous ATPases (A, A’components) which resemble the nucleotide binding domains in ABC transporters. Staphylococcus aureus biotin transporter (SaBioMNY) belongs to the subgroup II ECF transporters which are characterized by their shared use of energy coupling module (AT module) by several S components, with each having a different substrate preference. Therefore, characterizing the S families in ECF transporters are important for us to gain new knowledge about the mechanism of subgroup II ECF transporters. Besides, laboratory has developed a series of biotin analogues with antibacterial activity against S. aureus. Previous studies have demonstrated that these compounds were capable of binding to the S component of S. aureus(i.e.SaBioY). It was reasonable to speculate that these biotin analogues were transported across the S. aureus cells by the biotin transporter BioY. To further improve the antibacterial potency and selectivity, the binding and translocation mode of these compounds across the bacterial membrane via SaBioY needs to be defined. By utilizing a filter disk diffusion assay, I determined that the susceptibility of E. coli BL21 to antibiotics (erythromycin, streptomycin and chloramphenicol) was significantly increased when wild type SaBioY was heterologously overexpressed in the cells. A library of SaBioY mutants was also screened in this assay and the overexpression of all the mutants surprisingly increased the sensitivity of E.coli cells to all three antibiotics compared to the un-induced one. One exceptional mutant was the D157K/K160E that was able to restore the tolerance of cells to the antimicrobial agents. I reasoned recombinant SaBioY adopted a functional channel in the membrane of E. coli for low molecular weight antibiotics to diffuse through. In addition, I also found that R75, D157 and K160 are essential to the surrogate transport pathway since a single amino acid change can dramatically alter the sensitivity of E. coli cells to antibiotics compared to the wild type one. To further characterize the biotin core transporter SaBioY, I attempted to purify recombinant SaBioY from E. coliBL21 (DE3). The optimized conditions for expressing SaBioY were determined to be 1) culturing cells at 25°C, 2) using the richer potassium buffered TB growth medium and 3) using a high concentration of IPTG (0.8 mM). I have also developed a system for the scalable purification of this integral membrane protein using SDS as a solubilizer. 9.7mg of SDS-solubilized SaBioY (with expected molecular weight of 19,492 Da) was obtained from 2 liters of culture after IMAC purification, with 90% purity determined by Commassie staining gel. A small panel of available mild detergents was subsequently tested for their efficiency of extracting membrane protein from natural lipids with TrionX-100 giving the best extraction efficiency. This present study paves the way for further detergent screening and purification of SaBioY.
Thesis (M.Phil.) -- University of Adelaide, School of Molecular and Biomedical Science, 2014

The clinically relevant human pathogen Staphylococcus aureus employs an energy coupling factor (ECF) transporter to import the important micronutrient biotin. Like the well characterised ABC transporters, the ECF transporters utilise the hydrolysis of ATP to move substrates across biological membranes. However, the ECF transporters do not use a solute-binding protein to bind substrate instead employing a membrane embedded protein to fulfil this role. In certain bacteria, the substrate binding protein required for binding biotin is known as BioY. The aim of this thesis was to investigate protein structure and function relationships involving the BioY protein from S. aureus (SaBioY). S. aureus has functional import and export processes that result in a biphasic profile of biotin uptake. Active translocation of biotin was temperature-dependent, optimum at 30 minutes, and inhibited by biotin or structural analogues of the vitamin. The study demonstrated that recombinant SaBioY could be expressed in E. coli, and was localised to the membrane fraction as observed by Western blot analysis on fractionated cell lysates and fluorescence microscopy. Importantly, a convenient ligand binding assay was developed that facilitated deeper analysis of the SaBioY structure and function. SaBioY primarily recognises the ureido ring of biotin for substrate capture, but an intact thiophene ring also aids binding. Although a variety of functional groups can be appended onto the carboxyl group of the biotin moiety, the linker used to connect the molecules and the chemical property of functional group can impact binding to SaBioY. This knowledge can be exploited for developing biotin-based analogues with applications in antibiotic drug discovery. Since an X-crystal structure of SaBioY is not available, membrane topology predictions and computational modelling were used to generate a molecular module of SaBioY. This yielded a model containing 5 transmembrane domains, 3 extracellular loops, and 1 intracellular loop with intracellular N- and C-termini. Whilst the model was in good agreement with known crystal structures of other known S components, it possessed an additional V-shaped membrane embedded helix. Conserved amino acid residues in BioY were identified using the web-based Clustal-W alignment program and then mapped onto the SaBioY model. A series of 24 SaBioY mutants were then generated using random and site-directed mutagenesis approaches. Fluorescence polarisation based competitive-binding assays using a fluorescent-biotin tracer revealed several conserved (R75, D157 and K160) and non-conserved (N38, T54, F81, F88 and D128) residues important for biotin binding. Interestingly, a double mutant D157K/K160E completely abolished biotin binding. A filter disk diffusion assay using a panel of antibiotics showed recombinant expression of SaBioY increased E. coli antibiotic sensitivity to streptomycin, erythromycin and chloramphenicol, probably by forming a pore through channel by dimerisation requiring a dynamic cooperative interaction. Expression of all of the SaBioY mutants increased sensitivity to the three antibiotics. The D157K/K160E double mutant was an exception, as it had no effect upon antibiotic sensitivity. We proposed that D157 and K160 together play an essential role in SaBioY activity. In conclusion, this study successfully characterised the SaBioY transporter in both its native state and using a recombinant E. coli expression system. Substrate specificity of the transporter was determined, as was the channel gating potency of SaBioY for certain antibiotics when expressed in E. coli. Computational modelling and a novel FP based competitive assay also provided useful tools for biochemical analysis of SaBioY structure and function relationships. Further studies are now required to determine the SaBioY X-ray crystal structure, transport mechanism and regulation as well as to explore possible application of the transporter as a novel drug target or an alternative gating system new antibiotic agents.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2015.

Biotin protein ligase (BPL) catalyses the ordered reaction of biotin and ATP to give biotinyl-5’-AMP 1.03, which then activates a number of biotin dependent enzymes that are critical to cell survival. Research undertaken in this thesis highlights strategies to selectively inhibit Staphylococcus aureus biotin protein ligase (SaBPL) over the mammalian equivalent using 1,2,3-triazole and acylsulfonamide isosteres to replace the phosphoroanhydride linker found in biotinyl-5’-AMP 1.03. Chapter one describes the structure and catalytic mechanism of the target enzyme SaBPL, along with an overview of chemical analogues of biotin and biotinyl-5’-AMP 1.03 as BPL inhibitors reported to date. Preliminary studies on the utility of a 1,2,3-triazole as a bioisostere of the phosphoroanhydride linker of biotinyl-5’-AMP 1.03 are also discussed. Chapter two further examines 1,2,3-triazole analogues of lead SaBPL bisubstrate inhibitors 1.22 and 1.23. Specific chemical modifications were carried out at the ribose of biotinyl-5’- AMP 1.03, and a new class of purine analogues was developed to mimic the adenine group as in 1.03. In silico docking experiments using our x-ray structure of SaBPL aided in the design of these analogues by predicting optimal binding conformations. A structure activity relationship for the ribose and adenine mimics was developed and this revealed limited improvement in potency against SaBPL on modification at these two sites. Chapter three reports the first examples of truncated 1,2,3-triazole based BPL inhibitors with a 1-benzyl substituent designed to interact with the ribose binding pocket of SaBPL. In silico docking studies using a crystal structure of SaBPL aided in the selection of benzyl groups that present in the ribose-binding pocket of SaBPL. The halogenated benzyl derivatives 3.20, 3.21, 3.23 and 3.24 provided the most potent inhibitors of SaBPL with the respective Kᵢ value of 0.28, 0.6, 0.39 and 1.1 μM. These compounds also inhibited the growth of S. aureus ATCC49775 (MIC = 4 – 16 μg/ml), while possessing low cytotoxicity against HepG2 cells. Chapter four builds upon the active 1,2,3-triazole based inhibitors of SaBPL described in chapter two and three with an investigation at C5 of the triazole ring to generate 1,4,5- trisubstituted 1,2,3-triazoles. A class of 5-iodo 1,2,3-triazoles was synthesised from 1- iodoacetylene 4.02 and azides using CuAAC. Subsequent halogen exchange reaction allowed conversion of iodide to other halogens. 5-Fluoro-1,2,3-triazole 4.07, the lead compound from this series of inhibitors, proved to be a potent and selective inhibitor of SaBPL (Kᵢ = 0.42 ± 0.06 μM) and it significantly reduced S. aureus growth with no cell growth apparent at 16 μg/mL. Chapter five investigates the use of acylsulfonamide as a bioisostere of the phosphoroanhydride linker as in biotinyl-5’-AMP 1.03. Acylsulfonamide 5.05 was found as the most active and selective inhibitor of SaBPL (Kᵢ = 0.72 x 10⁻³ μM) and MtbBPL (Kᵢ = 0.74 x 10⁻³ μM) reported to date. Antibacterial studies revealed that 5.05 was active against susceptible S. aureus (MIC = 0.5-1.0 μg/mL), methicillin-resistant S. aureus ((MIC = 0.5- 1.0 μg/mL) and Mycobacterial tuberculosis ((MIC = 51 μg/mL). Finally, the x-ray structure 5.05 bound to SaBPL was solved to reveal important molecular interactions critical to the potency of 5.05 and emphasized the acylsulfonamide moiety as an effective bioisostere of phosphoroanhydride linker. Chapter six discusses the use of in situ click chemistry as an alternative approach for the synthesis of 1,2,3-triazoles. The target enzyme SaBPL was directly involved in the selection of its optimum triazole based inhibitor by catalysing the reaction of biotin acetylene and organic azides without copper as a catalyst. The use of high throughput LC/MS provided improved efficiency and sensitivity of detection of triazole-based inhibitors and allowed the in situ approach to be widely applied to BPLs from other bacteria. Chapter seven details the experimental procedures for compounds described in chapter 2 – 6, and the chromatographic analysis of in situ click experiments described in chapter 6.
Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2016.

There is an urgent need to discover new antibiotics to combat the rise of antibiotic resistant bacteria, such as methicillin resistant Staphylococcus aureus (MRSA). Many of the antibiotics currently in clinical use are synthetic derivatives of chemical scaffolds identified over 50 years ago in the golden era of antibiotic drug discovery. These antibiotics are often subject to existing resistance mechanisms and, as such, represent a short term solution to the antibiotic resistance crisis. Therefore it is imperative that new classes of antibiotics are developed that exhibit new modes of action and that are not subject to existing resistance mechanisms. Most antibacterial discovery efforts are focussed on drug targets with no mammalian equivalent. These targets have been well explored and therefore new antibacterial targets need to be identified. One strategy to identify new antibiotics is to explore targets that have a closely related human homologue. However, it is important that such inhibitors exhibit extremely high selectivity for the bacterial target over the human equivalent. One example of such a target is the essential enzyme biotin protein ligase (BPL) which catalyses the attachment of the micronutrient biotin onto biotin-dependent enzymes. In bacteria biotin-dependent enzymes play important roles in fatty acid synthesis and the tricarboxylic acid cycle. Without protein biotinylation these enzymes are devoid of activity and unable to perform their essential metabolic functions. Hence, inhibitors of BPL with selectivity over the human homologue represent a potential new class of antibiotic to combat MRSA. Our group has previously reported the X-ray crystal structure of S. aureus BPL (SaBPL) that provides the essential information necessary for structure guided design of new inhibitors. Of particular importance are two adjacent binding sites for the ligands biotin and ATP which, when bound, conjugate to form the adenylated reaction intermediate, biotinyl-5ʹAMP. Whilst amino acid residues in the biotin-binding pocket are highly conserved, residues in the ATP binding pocket are more variable and can be exploited to create species selective inhibitors. Our laboratory has previously reported analogues of biotinyl-5ʹAMP as BPL inhibitors where the labile phosphoanhydride linker present in the native reaction intermediate has been replaced with a non-hydrolysable 1,4-disubstituted-1,2,3-triazole linker. The triazole linker can be readily synthesised by the Huisgen cycloaddition reaction that occurs between an acetylene and azide. This cycloaddition reaction can proceed in two ways. Firstly, copper or ruthenium catalysts can be used to produce the 1,4 or 1,5 regio-isomers respectively. Alternatively, in special cases, this reaction can be catalysed by an enzyme. This is known as in situ click chemistry. Our laboratory has identified a biotin triazole pharmacophore, containing the biotinyl moiety and a 1,4-disustituted triazole. Various groups that can probe available binding sites on SaBPL can be conjugated to the triazole through click chemistry. The most potent triazole inhibitor of SaBPL, BPL068, had an inhibition constant of 90 nM and, importantly exhibited >1000-fold selectivity over the human homologue (Soares da Costa et al, Journal of Biological Chemistry, vol. 287, p 17823-17832). Here, the biotin triazole was conjugated to a 2-benzoxalone moiety that was designed to bind in the ATP binding pocket. This compound inhibited growth of S. aureus and did not show any in vivo cytotoxicity against cultured mammalian cells. Although BPL068 exhibited antibacterial activity, the effect was not strong enough to determine a minimal inhibitory concentration (MIC), which is required for a pre-clinical candidate. The first aim of this project was to characterize new SaBPL inhibitors with the goal of improving the antibacterial activity of the parent compound. Here I have employed structure guided drug design and protein biochemistry techniques to design new SaBPL inhibitors with desirable properties for pre-clinical candidates. To facilitate the characterization of SaBPL inhibitors I developed a high-throughput enzyme assay to measure protein biotinylation, and a surface plasmon resonance assay to determine the kinetics of ligand binding (Chapter 4). With these techniques in hand I have characterised 40 rationally designed SaBPL inhibitors. Biotinol-5ʹAMP, a literature compound that has previously been developed as a research tool to characterize BPL function, was first characterized. Here the inhibition of BPLs from a panel of clinically important bacteria was measured using an in vitro protein biotinylation assay. The spectrum of whole cell antibacterial activity was also addressed, with S. aureus and Mycobacterium tuberculosis being most susceptible to this compound (Chapter 5). A series of triazole inhibitors of SaBPL, designed to probe the ribose binding pocket was also investigated. Here 25, 1,4-triazole based compounds with 1-benzyl substituents were synthesized and tested for inhibition of SaBPL. These compounds are smaller in molecular weight compared to the parent molecule, BPL068, allowing further optimization by extending into the ATP binding pocket. The most potent compound from this series had an inhibition constant of 280 nM and exhibited antibacterial activity against S. aureus. Furthermore, all compounds did not inhibit the human homologue or cultured mammalian cells (Chapter 6). A further series of compounds were next synthesized to optimise the triazole linker in BPL068. Firstly, compounds were synthesized in which the triazole linker has been replaced with alternative heterocycles with a view to improving its biological activity. A 1,2,4-oxadizole linker was found to inhibit SaBPL with an inhibition constant of 1.2 μM, with no inhibition of the human homologue (Chapter 7). A separate series of 1,4,5-trisubstituted-1,2,3 triazole analogues were also investigated (Chapter 8). Here, the hydrogen of the C5 atom in the triazole heterocycle was replaced with halogenated substituents to investigate whether halogenation of BPL068 could improve antibacterial activity. A 5-fluoro-1,2,3 triazole was found to inhibit SaBPL with an inhibition constant of 420 nM. Importantly, the fluorinated analogue exhibited an MIC of 8 μg/mL against a clinical isolate of S. aureus. This compound is the first example of a triazole based BPL inhibitor in which an MIC could be determined. Following the identification of BPL antibacterials, the mechanism of action needed to be addressed. Therefore the second aim of my project was to develop probes that could facilitate mechanism of action and uptake studies. Here a fragment based approach was employed using in situ click chemistry. In situ click chemistry relies on the ability of the target enzyme to select out and synthesize its own inhibitors from a series of small molecule building block precursors. This technique exploits the Huisgen cycloaddition reaction that proceeds between an acetylene and an azide to produce the 1,2,3-triazole heterocycle. Here, I have demonstrated that the in situ click chemistry approach could be adopted to identify inhibitors of a panel of 4 BPLs from clinically important bacteria. Next, azide-functionalized analogues of 2 fluorophores were synthesized and tested for chemical ligation to biotin acetylene using BPL as a catalyst. The ‘clicked’ compounds were confirmed for inhibition of SaBPL and entry into S. aureus. This newly developed probe will be used in solution based and con-focal microscopy studies to probe the mechanism of entry and action in S. aureus (Chapter 9). In summary, I have characterized a new series of SaBPL inhibitors that have improved antibacterial activity and still maintain the selectivity required for a pre-clinical candidate. Using in situ click chemistry, I also developed a new inhibitor that will be used to probe the mechanism of entry and action of BPL inhibitors in S. aureus. The work demonstrated in this thesis will be used to help optimize BPL inhibitors, ultimately leading to the development of a pre-clinical candidate.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Biological Sciences, 2017.

Staphylococcus aureus is a versatile and potentially dangerous human pathogen. One of the traits of S. aureus that is crucial for its survival during pathogenesis is its ability to quickly adapt to changes in the microenvironment, including an ability to adapt to the limited availability of micronutrients such as biotin. Biotin is a co-factor required for important metabolic enzymes such as pyruvate carboxylase (PC) and acetyl CoA carboxylase (ACC). In certain bacteria like S. aureus, the protein that is responsible for managing biotin homeostasis is the biotin retention protein, BirA (also known as biotin protein ligase or BPL). BirA is a bi-functional protein that serves as both the enzyme responsible for protein biotinylation and a transcriptional repressor that regulates biotin biosynthesis and import. Escherichia coli BirA (EcBirA) has been well studied, however, less extensive studies have been performed on S. aureus BirA (SaBirA). Whilst EcBirA regulates transcription of the biotin biosynthesis operon (bioO), SaBirA has multiple targets including bioO, the biotin transporter (SabioY) and genes involved in fatty acid synthesis (SayhfS-SayhfT). For both EcBirA and SaBirA, homodimerization is a pre-requisite for DNA binding and subsequent repressor activity. In the absence of protein requiring biotinylation, and when cellular demand for biotin is low, BirA will dimerize, bind to its target DNA and repress expression of biotin biosynthetic enzymes. Previous studies in our laboratory revealed clear differences between EcBirA and SaBirA. One of these differences is that dimerization and DNA binding of EcBirA only takes place when the protein is in complex with the reaction intermediate biotinyl-5ʹ-AMP (i.e. the holo-enzyme), whereas SaBirA was able to dimerize and bind DNA in both the holo (KD²⁻¹= 29 μM, KD DNA = 108 nM) and non-liganded (i.e. apo) states (KD²⁻¹ = 30 μM, KD DNA = 649 nM). I hypothesized that there are clear distinctions in the DNA binding interaction between SaBirA and the well-studied EcBirA. These differences allow S. aureus to elegantly orchestrate biotin synthesis and transport in response to external biotin availability. This study aims to define SaBirA-regulated gene expression using in vitro and in vivo methods. In addition, the effect of extracellular biotin concentration on biotin uptake and gene expression in both S. aureus and E. coli were also investigated in this study. The result showed that within 30 minutes, biotin starved S. aureus could sense changes in exogenous biotin and responded with increased biotin uptake and down regulation of biotin synthesis (>100-fold). These rapid responses were not observed in E. coli. Furthermore, the DNA-binding activity of SaBirA was also probed in vivo. Since S. aureus is not naturally competent to transformation, it can be technically difficult to genetically manipulate this bacteria. To overcome this problem, reporter strains were constructed in E. coli containing chromosomally integrated SaBirA and EcBirA, as well as their target promoters fused to a lacZ reporter gene. Here I confirmed that birA from both bacteria are biotin-responsive transcription factors. Moreover, based on the dimerization constant of apo- SaBirA (KD²⁻¹ = 30 μM) and apo-EcBirA (KD²⁻¹ = 2 mM), and the predicted intracellular concentration of BirA (2nM – 100nM), it is estimated that these apo proteins are predominantly monomeric in growing cells. Therefore, mutant proteins with abolished in vitro dimerization ability were included as mimics of the monomeric apo-state, namely SaBirA F123G and EcBirA R119W . The results obtained from the in vivo assays showed that SaBirA F123G repressed the target promoters, whereas EcBirA R119W was devoid of repressor activity. These results were confirmed in vitro by gel-shift assays. Cross-linking studies added further evidence that DNA promotes dimerization of SaBirA F123G, but not E. coli R119W. In vitro analysis also revealed the affinity for DNA binding varies between SaBirA-target promoters. This suggested a hierarchy of SaBPL regulated genes, with the biotin biosynthesis operon being the most responsive to exogenous biotin concentration. Taken together, the outcomes from in vivo and in vitro analyses performed in this study have validated the hypothesis that SaBirA uses different DNA binding mechanisms to EcBirA. As a consequence, SaBirA provides S. aureus with one avenue to adapt in response to its environment. Finally, this study also investigated the role of a novel SaBirA inhibitor, BPL199, as a co-repressor in DNA binding and its effect on gene transcription. Quantitative Real-Time PCR experiments revealed that BPL199 was able to act as a co-repressor to down-regulate expression of biotin-regulated genes in vivo, with similar kinetics as biotin. EMSA analysis showed that the affinity of SaBPL:BPL199 for DNA binding was similar to that of the natural substrate, biotinyl-5ʹ-AMP. This supported the proposal that BPL199 successfully mimics the action of biotinyl-5ʹ-AMP in initiating transcriptional repression. In addition, a BPL199-resistant strain of S. aureus generated in our laboratory, was also investigated. DNA sequencing revealed a single point mutation in SaBirA (D200E) that mapped within its dimerization interface. The ability of SaBirA D200E to bind DNA, and down regulate gene expression, was subsequently addressed. The results indicated that SaBirA D200E was compromised in the SaBirA:DNA interaction in vivo. The most susceptible target was the SabioY promoter, suggesting that increased transport of exogenous biotin is one mechanism that can be employed by the bacteria to overcome compounds that target BPL. Thesis layout: The thesis will be presented as a combination one published literature review, one manuscript to be submitted for publication as well as conventional chapters. Each manuscript will be a chapter with its own references. A general introduction and discussion will also be included to link together all the research conducted during this candidature. A publishing agreement with all co-authors involved with the work is also included.
Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2017

There is a well-documented need to replenish the antibiotic pipeline with new products to combat the rise of drug resistant bacteria, such as the superbug methicillin resistant Staphylococcus aureus (MRSA). One strategy to combat drug resistance is to identify new chemical classes with novel mechanisms of action and that are not subject to existing resistance mechanisms. As most of the obvious bacterial drug targets with no equivalents in mammals have been well explored, targets with a closely related human homologue represent a new frontier in antibiotic discovery. However, to avoid potential toxicity to the host, these inhibitors must have extremely high selectivity for the bacterial target over the human equivalent. This thesis is focused upon exploiting the ubiquitous enzyme biotin protein ligase (BPL), which is involved in the essential cellular process of attaching biotin onto biotin-dependent enzymes. Due to the pivotal metabolic roles played by biotin-dependent enzymes in bacteria, BPL has been proposed as a promising new antibiotic target. Hence, BPL inhibitors with selectivity for the bacterial isozyme over the human equivalent promise a new class of antibiotic to combat MRSA. The aim of this project was to provide proof of concept data demonstrating that BPL from a pathogen could be selectively targeted for inhibition over the human equivalent. Here I employed a combination of structure-guided drug design and fragment-based approaches to discover novel BPL inhibitors. The X-ray crystal structure of S. aureus BPL (SaBPL) shows two adjacent binding sites for the ligands biotin and ATP, making it an ideal candidate for a fragment-based approach to drug discovery. Although the residues at the biotin-binding site are highly conserved, the nucleotide pocket shows a high degree of variability that can be exploited to create compounds selective towards BPLs from pathogens. The biotin 1,2,3 triazole analogues identified in this work yielded our most potent and selective inhibitor (Ki = 90 nM) [i is subscript] with >1100-fold selectivity for the SaBPL over the human homologue (Chapter 2). The molecular basis for the selectivity was identified using mutagenesis studies with a key arginine residue in the BPL active site necessary for selective binding. Importantly, the biotin triazole inhibitors showed in vivo cytotoxicity against S. aureus, but not cultured mammalian cells (Chapter 2). In an attempt to identify new chemical scaffolds with improved ligand efficiency for chemical development, a series of analogues based on the natural ligand biotin were also designed and tested for enzyme inhibition and antimicrobial activities against clinically relevant strains of S. aureus (Chapter 3). This approach resulted in highly potent compounds (Ki < 100 nM) [i is subscript] with antibacterial activity against MRSA strains (MIC = 2 – 16 μg/mL). Whilst only moderate selectivity over the human enzyme (10 - 20 fold) was observed, the biotin analogues provided a suitable chemical scaffold with high ligand efficiency for further chemical development. One of the compounds identified was biotin acetylene, which forms a long lived complex with SaBPL and is a precursor for in situ click reactions. This target-guided approach to drug discovery relies on the ability of the enzyme to choose its own inhibitors from a range of acetylene and azide building blocks to form specific triazole products. Since a class of biotin-triazole molecules had already been identified as selective inhibitors of SaBPL, we reasoned that this enzyme would provide an ideal candidate for performing in situ click approach to inhibitor discovery. In this work, a protocol for the BPL-catalyzed in situ click reaction was optimized to select the optimum triazole-based inhibitor using biotin acetylene as an anchor molecule to recruit complimentary fragments that could bind in the peripheral ATP pocket (Chapter 4). The in situ reaction was shown to be improved by the use of a SaBPL mutant that promoted diffusion of the triazole product from the active site following synthesis. This novel approach improved efficiency and ease of detection (Chapter 4). Apart from drug discovery, this thesis also focuses on enzymatic characterization of SaBPL and highlighting the key differences between SaBPL and the human homologue. The structure of human BPL is yet to be reported, so structure-function studies were performed to elucidate new information about the bacterial and human enzymes. The oligomeric state of SaBPL was investigated using analytical ultracentrifugation in its apo form and in the presence of ligands (Chapter 5). Unlike human BPL, SaBPL was shown to dimerize in solution. A single amino acid in SaBPL, Phe123, was identified to have a dual key role in dimer formation and inhibitor binding (Chapter 5). One of the major roadblocks to obtaining crystals of the full-length human enzyme is the low yield of protein obtained from recombinant expression and purification. In this thesis, an alternative approach is described that could be used to increase our chances of obtaining structural data about the human BPL. I created a ‘humanized’ chimeric protein in which all seven residues in the nucleotide pocket of SaBPL that are not conserved with the human BPL were mutated to their human equivalents. This ‘humanized’ protein exhibited similar kinetic and inhibition properties to the human enzyme (Chapter 6). Crystal trials have commenced to help direct future drug development efforts. Further studies on the human BPL enzyme will also be described, including the dissection of the binding mechanism using surface plasmon resonance (Chapter 7). The N-terminal domain of this enzyme was shown to play a role in stabilizing the complex between the enzyme and the biotin domain substrate, providing the first molecular explanation for human BPL-deficient patients that do not respond to biotin therapy. In summary, this work demonstrates for the first time that BPL from the clinically important pathogen Staphylococcus aureus can be selectively inhibited. A provisional patent has been filed for the biotin 1,2,3 triazole molecules I have identified. These discoveries will enable further development of a new class of antibiotics.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2013

碩士
國立中興大學
生命科學系所
94
Cadmium(Cd) and lead(Pb) are widely used in industrialized countries, and both are non-essential heavy metals for the organisms. When they are accumulated, they will become extremely toxic to living organisms. For instance, the Minimata disease caused by mercury contamination, and the Itai-itai disease caused by cadmium contamination had made serious injury to the biological system for its bioaccumulation in the food chain. These heavy metals could be a threat at the low levels. Therefore, mechanisms responsible for minimizing the concentraction of non-essential heavy metals is required for all organisms. This study was initiated to clone and characterise of cadA gene originated from cadCA operon in a gram-positive bacteial plasmid, Staphylococcus aureus plasmid pI258, into Arabidopsis thaliana.The expression of cadA gene in A. thaliana may increase its resistance to cadmium, lead and zinc(Zn) and decrease the heavy metal content in the transgenic plants. The isolated transgenic lines were conferred both in DNA and RNA levels. Analysis of transgenic A. thaliana plants expressing cadA showed that CadA is functionally active and that the plants have enhanced resistance of Cd(II), Pb(II) and Zn(II), while accumulated a greater amounts of Cd(II) or Pb(II). These results suggest that transgenic plants expressing cadA may be an useful tool for phytoremediation.